Targeting the Highly Conserved gp41 MPR Neutralizing Determinant We are pursing 3 distinct means to elicit neutralizing antibodies directed against the gp41 MPR. One is the expression of MPR miniproteins captured and displayed on the surface of solid phase proteoliposomes. The second approach utilizes the highly immunogenic HepB surface antigen nanopaticles to display the MPR in selected contexts. The third approach we are pursuing is to stabilize the 2F5 epitope on heterologous protein scaffolds in collaboration with Bill Schief and David Baker at the University of Washington and Peter Kwong at the VRC. For this approach, the extended loop 2F5 epitope is precisely scaffolded onto structurally defined non-HIV proteins available in the protein data base. Once transplanted, the 2F5 epitope-scaffold will be tested for binding and crystallized (by the Kwong lab). The plan is to generate three to four scaffolds generated from the structural modeling that bind 2F5 with nanomolar affinity. Once produced, the 2F5 scaffolds will be crystallized in the free-state (without 2F5 present). Those that closely fit the 2F5 antibody-induced conformation will be tested for immunogenicity to determine their ability to generate 2F5 epitope specific responses and of course to determine if they elicit neutralizing antibodies. We will test the 2F5 protein scaffolds individually or in prime:boost combination to focus the immune response on the unique and common 2F5 epitope fold. The 2F5 epitope scaffold proteins will also be tested in prime:boost combination with the gp160 PLs or expressed in the HepB particulate context. For any of the MPR-directed approaches we will use assess if heterologous T help is required to enhance immunogenicity or if toll-like receptor (TLR) ligand adjuvants will better elicit antibody responses against the potentially self resembling determinants 13. We also plan to test a selected set of constructs in autoimmune strains of mice to determine if the cardiolipin-like antibodies commonly elicited in these animals might be a beneficial response to then drive to affinity maturation with the MPR immunogens. I. Min Tang has several projects ongoing that relate to the MPR and to improvements of the Env PLs as immunogens and prime-boost strategies with the PLs, and has been invaluable using several expression systems to generate selected cores for structure. Also she has done some cellular assays which may have some value in interpreting immune response to different immunogens. a. Immunogenicity to determine if we can elicit antibodies against the gp41 MPR employing membrane various issues are being tested;is the MPR DNA immunogenic, MPR DNA+PADRE (heterolgous T cell help since we are not sure that the short MPR sequence contains a helper epitope), MPR PLs +PADREin combination with prime-boost of EnvPL gp160. b. Sequential boosting of EnvPL in order to drive what is commonly conserved between these clade isolates (CD4BS, gp41 MPR) and not the variable region-directed response. c. We have conducted studies including MPL or T helper lipopeptides into the PLs to enhance their immunogenicity. We have also changed lipids (more similar to the viral membrane composition) to enhance 2F5/4E10 binding. II. Sanjay Phogat was working with the Hepatitis B surface antigen which form 22nM particles to present the MPR for binding and immunogenicity analysis. Production of high-levels of pure particles from baclovirus expression and yeast expression were accomplished and data published. Phogat S, Svehla K, Tang M, Spadaccini A, Muller J, Mascola J, Berkower I, Wyatt R.Analysis of the human immunodeficiency virus type 1 gp41 membrane proximal external region arrayed on hepatitis B surface antigen particles.Virology. 2008 Mar 30;373(1):72-84. Epub 2007 Dec 26 III. Javier Guenaga is examining the 2F5 epitope as defined by the Ofek-Kwong structure locked onto selected protein scaffolds (in collaboration with David Baker) to be examined by production, binding, immunogenicity and structure. Since there seems to be an indication that prime-boost, coupled with cysteine-stabilization actually works, the likelihood of significant outcome has increased. Future activities within this project include cloning, bacterial and mammalian expression, refolding, ELISAs/Biacore, neutralization assays and integrating structural information into enhanced immunogen design. A manuscript is in preparation on the original scaffold design and characterization.